Current techniques used in the quality control of culture media required for pharmaceutical microbiology

Cara N. Wilder, Ph.D.

Culture media is a basic tool of microbiology, supporting most microbiological assays such as propagation, obtaining pure cultures, enumerating cells, preservation, and selecting microorganisms. For pharmaceutical quality control laboratories, media is frequently used for environmental monitoring, sterility testing, and microbial enumeration tests. As such, the quality of microbial-based tests greatly depends on the quality of the culture media; if the media is not the right quality, it undermines all of the tests that the media is used for. Therefore, safeguarding the quality of culture media through routine quality control testing can help ensure reliable, consistent microbiological test results.

Culture media are traditionally defined as substances that encourage the growth, support, and survival of microorganisms. This is achieved through the preparation of liquid broth or solid agar media comprising reagents required to support microbial growth, including basic nutrients, energy sources, growth factors, minerals, buffer salts, and metals¹. To help minimize lot-to-lot variability, culture media manufacturers often attempt to standardize the preparation of media; however, there are frequently unavoidable differences in raw materials from natural sources, intra-lab skill level, or the storage and shipping conditions of the media. To help counter this, culture media should be physically inspected for color, clarity, damage, pH, and gel strength following all preparation steps. Moreover, media that has not been quality control tested or assessed should be quarantined to prevent premature use.

In addition to the physical inspection of culture media, it is important to analyze media for microbiological characteristics. As these media are frequently used for the analysis of sterility throughout the manufacturing process, or for the detection of objectionable microorganisms, it is imperative that the media is not only sterile, but able to promote the growth of any possible contaminants. For the analysis of media sterility, uninoculated media is commonly incubated. Here, the growth temperature and incubation period will depend on the type of media being analyzed. Media exhibiting the absence of growth following the recommended incubation period are considered sterile. In contrast, growth promotion testing is one of the most important quality control tests performed on media and is used to determine if the media in question is able to promote and sustain growth. The primary objective of the test is to detect if a new batch of media is functional and of the same standard as the most recent batch of media tested, as well as ensures the consistent use of standardized media between labs.

Currently, there are several approaches that can be taken for testing media for growth promotion. For agar-based media, a simple method of analysis is to serially dilute microbial strains and plate them on test media using the spread plate technique. These agar plates are then compared to the growth characteristics of a control plate, which is a batch of media that has been previously assessed for growth promotion and approved for use. Two other, more robust, approaches include the Miles-Misra and the ecometric techniques². The Miles-Misra method is a quantitative technique used to determine the surface viable count. Here, droplets of titered microbial suspensions are dropped onto plates and allowed to spread naturally; the test plate is compared to a control plate following incubation. Colonies are counted in the sector where the highest number of discrete colonies can be enumerated. The results of the assay are examined using a productivity ratio, which is equivalent to the mean of the test plates divided by the mean of the comparative control plates. In this case, an acceptable productivity ratio should fall within 0.5-2, which is equivalent to 50%-200% recovery. In contrast, the ecometric method is a semi-quantitative approach that involves streaking a loopful of a microbial suspension onto four quadrants of an agar plate so that the inoculum is diluted with each streak. In this method, growth should occur in each of the quadrants.

For liquid broth culture media, growth promotion testing typically involves inoculating the media with an estimated number of microorganisms (< 100 colony forming units) and observing it for turbidity within a required time. For bacterial and fungal cultures, this is typically 3 and 5 days, respectively. These inoculated test cultures are compared to control batch media, and the inoculum level is verified via plate count. Both the test media and the control media must show turbidity for the analysis to be considered successful.

For each media type, an appropriate panel of microorganisms is required in order to demonstrate the suitability of the media for the required test. Depending on the required use of the media, a suitable microbial panel may differ in the microbial strains selected as well as how many strains comprise the panel. Generally, the pharmacopeia recommends a preselected list of specific microorganisms for each chapter or general test that must be traceable to a reputable culture collection, such as ATCC. For example, a standard set of cultures may include Staphylococcus aureus subsp. aureus (ATCC^® 6538™), Bacillus subtilis subsp. spizizenii (ATCC^® 6633™), Pseudomonas aeruginosa (ATCC^® 9027™), Clostridium sporogenes (ATCC^® 19404™), Candida albicans (ATCC^® 10231™), Aspergillus brasiliensis (ATCC^® 16404™), Escherichia coli (ATCC^® 8739™), and Salmonella enterica subsp. enterica serovar Typhimurium (ATCC^® 13311™). In addition to the recommended set of cultures, isolates that are commonly found in the manufacturing environment are commonly used for media testing as well. For example, media used for the analysis of clean room sterility are often tested for the growth promotion of microorganisms commonly found in clean rooms, such as Staphylococcus, Corynebacterium, Micrococcus, Bacillus species, and common skin microflora.

ATCC Genuine Cultures^® are maintained using the seed lot system recommended by the United States Pharmacopeia (USP) General Chapter, Microbiological Best Laboratory Practices <1117>. Moreover, each ATCC Genuine Culture^® has been thoroughly authenticated and characterized using a polyphasic approach comprising genotypic and phenotypic analyses. To conserve the characteristics of ATCC Genuine Cultures^®, it is recommended that each laboratory has a seed lot system in place for preserving and maintaining reference cultures; these cultures must be handled carefully at all times to avoid genetic drift, phenotypic changes, contamination, and strain damage.

Overall, microbial culture media is an important tool used in the pharmaceutical quality control process. As the quality and functionality of the media directly affects its use in microbiological assays, it is imperative that it is thoroughly tested for quality, sterility, and growth promotion prior to its use. This can be analyzed through the use of fully characterized ATCC Genuine Cultures^®. All ATCC Genuine Cultures^® are maintained and authenticated in accordance with the USP Microbiological Best Laboratory practices, ensuring reliable results of microbial assays.


References

1.      Bridson E, Brecker A. Design and Formulation of Microbiological Culture Media. In J.R. Noris and D.W. Ribbons (Editors). Methods in Microbiology, Volume 3A, London: Academic Press, 1970.

2.      Mossel DAA, et al. Quality control of solid culture media: a comparison of the classic and the so-called ecometic technique. J Appl Bacteriol 49: 439-454, 1980.

The importance of authenticated quality control strains in supporting guidance for industry

Cara N. Wilder, Ph.D.

Section 510(k) of the Food, Drug, and Cosmetic Act requires device manufacturers to notify the Food and Drug Administration (FDA) of their intent to market a medical device. This process, known as Premarket Notification, allows the FDA to determine if an equivalent legally marketed device already exists, ensures that the new device is properly identified and classified, and that the device is cleared for use¹. For devices that pose a serious level of risk of illness or injury to the user, such as those that are used internally or to sustain life, a Premarket Approval (PMA) submission is required. This is the most stringent type of approval application required by the FDA, and requires information on how the medical device was designed and manufactured as well as any preclinical and clinical studies on the device. For a medical device to acquire PMA, it must be backed by sufficient valid scientific evidence assuring that it is safe and effective for its intended use.

As part of the requirements for 510(k) clearance or PMA, medical devices must be examined using appropriate FDA guidances; the recommended guidance documents depend on the classification and the intended use of the device. For example, for the development of a 510(k) in vitro diagnostic (IVD) device intended for the detection of Clostridium difficile, the FDA has issued a draft guidance entitled, “Draft Guidance for Industry and Food and Drug Administration Staff – Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection of Clostridium difficile.”² This particular guidance recommends various analytical, clinical, and cross-contamination studies for establishing the performance characteristics of IVDs developed for the detection of C. difficile in stool samples via antigen-, antibody-, or nucleic acid-based tests.

One of the key features of this guidance, and those similar to it, is the recommendation for determining the analytical sensitivity and cross-reactivity through the use of authenticated, characterized strains. For determining the analytical sensitivity of a C. difficile detection assay, the FDA recommends the use of a variety of strains that represent the various known C. difficile toxinotypes (0; IIIb; IIIc; tcdA- , tcdb-; V; VIII, XII, and XXII). To analyze cross-reactivity, the FDA recommends the use of medically-relevant viruses and bacteria of varying species such as Bacillus cereus, Citrobacter freundii, and Clostridium tetani.

To support the need for highly characterized strains, ATCC has fully authenticated and described microbial strains that are recommended in guidances for industry. For the aforementioned C. difficile guidance, ATCC offers a number of C. difficile strains that have been genotypically and phenotypically authenticated as well as functionally characterized for toxinotype, binary toxin, and ribotype. These defined characteristics, along with the provided isolation history, allows for the easy selection of strains recommended for testing the analytical sensitivity of novel medical devices. Moreover, the expansive breadth of the ATCC collection allows for the easy obtainment of representative strains for cross-reactivity testing.

ATCC similarly supports a number of other guidance documents, such as the “Draft Guidance for Industry and Food and Drug Administration Staff - Establishing the Performance Characteristics of Nucleic Acid-Based In vitro Diagnostic Devices for the Detection and Differentiation of Methicillin-Resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA)” ³. In this latter guidance, characterized S. aureus strains with known SCCmec type and PFGE type are needed for establishing analytical sensitivity, and pathogenic and commensal flora found in the nares should be tested to analyze cross-reactivity. To aid in the development of these diagnostic devices, ATCC has fully characterized a majority of the S. aureus strains in the collection for both SCCmec type and PFGE type, and have confirmed the presence of the mecA gene in methicillin-resistant strains.

Overall, when developing a novel medical device, it is important to ensure that the device is properly evaluated and verified based on FDA guidance recommendations prior to submitting it for 510(k) clearance or PMA. Using authenticated, fully characterized strains from an ISO accredited and certified standards development organization, such as ATCC, can help ensure the reliability and reproducibility of analytical sensitivity and cross-reactivity data, thus confirming the efficacy and validity of the device in question.

References

1. Food and Drug Administration. Premarket Notification (510k). Available online: http://www.fda.gov/medicaldevices/deviceregulationandguidance/howtomarketyourdevice/premarketsubmissions/premarketnotification510k/default.htm  
2. Food and Drug Administration. Draft Guidance for Industry and Food and Drug Administration Staff - Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection of Clostridium difficile. Available online: http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm234868.htm.
3. Food and Drug Administration. Draft Guidance for Industry and Food and Drug Administration Staff - Establishing the Performance Characteristics of Nucleic Acid-Based In vitro Diagnostic Devices for the Detection and Differentiation of Methicillin-Resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA). Available online: http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm237235.htm